JPH09511521A - Fusing liposome, method for producing the same and method for using the same - Google Patents

Abstract

(57) [Summary] (i) In addition to a neutral bilayer-forming lipid, a fusion-promoting effective amount of an ionizable lipid having a protonatable cation head group and an unsaturated acyl chain Provided is a liposome composition comprising a liposome having a lipid bilayer and (ii) a compartment adjacent to the outermost lipid bilayer containing an aqueous solution having a first pH. Outside the liposomes in this composition is an aqueous solution having a second pH. The first pH is less than the pK _a of the ionizable lipid in the outermost lipid bilayer and the second pH is greater than the pK _a of the ionizable lipid in the outermost lipid bilayer, and thus There is a pH gradient across the outer lipid bilayer, and depending on the gradient, ionizable lipids accumulate within the inner monolayer of the outermost lipid bilayer. Liposomes have neutralized ionizable lipids, are evenly distributed in the outermost lipid bilayer, and reduce the pH gradient when fusion occurs in a controlled manner such that they are fusogenic. It can be fused to other lipid bilayers, such as the plasma membrane of mammalian cells. Controlled fusion can be used to control the delivery of liposome-entrapped bioactive agents to cells.

Description

Detailed Description of the Invention           Fusing liposome, method for producing the same and method for using the same   This application is one of US Patent Application No. 985,673 filed on Dec. 4, 1992. Continuing application, fusogenic, enhanced fusion to other lipid bilayers ) Liposomes, method for producing the liposomes, and other lipid bilayers of the liposomes (Lipid bilayer) to a method for controlling fusion.   Liposomes are self-consistent membranes composed of one or more bilayers of amphipathic lipid molecules. Collective structures, each of which surrounds an internal aqueous volume. Lipid bisection The amphipathic lipid molecule that constitutes the child membrane is composed of one or two non-polar (hydrophobic) acyl chains. It contains polar (hydrophilic) headgroup regions that are covalently bound. Hydrophobic Between the water-soluble acyl chain and the aqueous solution surrounding the lipid molecule With no contact, the polar headgroups are oriented towards the aqueous solution, while the reed When the lipid molecules are rearranged so that the chain is oriented towards the interior of the bilayer, It is generally believed. The end result is two opposing monolayers. An energy-stable lipid bilayer structure consisting of Effectively shields contact with.   Liposomes can be prepared in various ways. Bangham's method (J. Mol. Blo., Vol. 13, 238-252 (1965)), "normal" muller. Multilamellar liposomes (MLV) are made. The "normal" MLV is , Can have an uneven solute distribution between its aqueous compartments, thereby Osmotic pressure occurs. Lenk et al. (US Pat. Nos. 4,522,803 and 5,030, No. 453 And No. 5,169,637), Fountain et al. (US Pat. No. 4,588,57). 8) and CulliS et al. (US Pat. No. 4,975,282) are those of an aqueous compartment. The distribution of solutes taken into each is substantially equal, that is, the solute distribution between lamellas is A method of making substantially equal multilamellar liposomes is disclosed. Practically equal Having a good interlamellar solute distribution means that the osmotic pressure between the aqueous compartments of these MLVs is high. Means less than, and thus is generally more stable than a regular MLV. I can say. Unilamellar liposomes are sonicated (Paphadjopoulos Et al. (1968)) or extrusion (Cullis et al., US Pat. No. 5,008,050). And Loughrey et al., US Pat. No. 5,059,421), manufactured by MLV. Can be built.   Liposomes contain a physiologically active agent passively, that is, a water-soluble agent. In some cases, by solubilizing the molecule in the medium in which the liposome is formed, Or by adding a lipid-soluble agent to the lipid solution in which the liposomes are prepared , Can be loaded. Ionizable bioactive agents also cross the liposome membrane To create an electrochemical potential gradient and then add the agent to the medium outside the liposomes Thus it can be loaded into liposomes (Bally et al., US Pat. No. 5,077, 056).   Drugs entrapped in liposomes can reduce toxicity or increase efficacy Or both can increase the therapeutic index. Furthermore, in the circulatory system Like other particulate matter, liposomes are found in tissues with sinusoidal capillaries. Are ingested by phagocytic cells of the reticuloendothelial system, which often leads to sites of intracellular infection. Directed to.   The fusion of biological membranes involves endocytosis, fertilization and intracellular transport of proteins. It is an important process in various cell transport functions including delivery. Liposome And cell fusion, the outermost bilayer of the liposome unifies with the plasma membrane of the cell (See Huang (1983)). Outermost for fusion to happen The lipids of the layer lipid bilayer must be mixed with the lipids of the plasma membrane of the cell. The mechanism proposed for the fusion between lipids is the charged headgear. Including non-bilayer-preferring structure This will effectively change the shape of the lipid species. These are the nucleation sites for membrane fusion Sequentially producing other defects in micelles or bilayers that act as And Hope (1978)).   Studies using liposomes have shown that the tendency of liposomes to fuse and the component lipids Square H₁₁Correlation between the tendency to take a non-bilayer like phase has been demonstrated, For example, non-bilayer structures such as inverted micelles or other bilayer defects can cause fusion events. It has been suggested that it is an intermediate structure in (for example, Cullis et al. (197). 8 years))). Lipid composition contributes to the tendency of liposomes to adopt non-bilayer structures. (Eg Martin and MacDonald (1976), Martin And MacDonald (1976), Weismann et al. (1997)). For example, negatively charged phospholipids (eg, phosphatidic acid (PA), phosphatidic acid (PA), A film containing fatidylserine (PS), etc.₊₊The existence of divalent cations such as It has been shown to fuse together (Hope et al. (1983)). Synthetic cation Lipids have been shown to fuse with high concentrations of negatively charged counterions (Duzgunes et al. (1989)). Unsaturated diglycerides are also influential fusogens (f usogen) (Das and Rand (1986)). Furthermore, the obi Non-charged lipids can more easily assume a non-bimolecular structure and thus their It is considered to be more fusogenic than the charged form (eg, Gruner et al. (19) 1985))).   Cationic lipids increase the efficiency of mammalian cell transfection Have been shown to be effective in increasing (eg, Malone et al. (1989) , Konopka et al. (1991)). These applications are encapsulated in liposomes Extended to the in vivo delivery of stored substances, the cytotoxicity and hemolysis caused by cationic lipids And the problem of increased coagulation response arises (Senior et al. (1991)). These shadows The sound seems to occur above the threshold level of positive charge and depends largely on the nature of the cation species. I can't think it exists. By synthesizing a new cation head group Attempts have been made to solve the toxic effects of cationic species, but for the most part Remains unresolved. There is a possibility that these lipids can be applied to the intended drug delivery. If so, the toxic side effects can be reduced by reducing the cation concentration. The ability of these compounds to control the in vivo fusogenicity is desirable.   Fusion between vesicle populations with opposite charges has also been demonstrated (Stam atos et al. (1988)). Control of liposomes using induced lipid asymmetry Previously announced fusion (Eastman et al. (1991) Eastma. n et al. (1992), Redelmeier et al. (1990). However, these are It uses anionic, ionizable lipids, which are The pH gradient used to deliver is to load the cationic drug into the liposomes. (See Bally et al., US Pat. No. 5,077,056). This The controlled fusion of these drug-loaded liposomes produces similar fusions. It will be better done with compatible cationic lipids.   Delivery of liposome contents to cells by fusion of liposomes and biological membranes Can be. The injection of the aqueous contents of liposomes into the cytoplasm is accomplished by Illustrated by fluorescence dequenching of cein (Weinstein  Et al. (1977), Huang et al. (1978)). Other reports include liposomes Bioactive substances (eg, cAMP, ricin, actino) Mycin D, Ca₊₊, MRNA, viruses and viral genomes), It was shown that it can be introduced into cells by fusion of mulipid bilayer and cell membrane. (Eg Paphadjopoulos et al. (1974), Dimitriadis and Butters. (1979), Theoharides and Douglas (1978), Ostro et al. (197). 8), Dimitriadis (1978), Wilson et al. (1979), Fraley et al. (1) 980)). However, in other studies, fusion has shown that liposomes interact with cells. It is suggested that it is not the main mechanism of action (eg Szoka et al. (19 80), Hagins and Yoshikami (1982), Pagano and Takeichi (1) 977))).   Although the above publications are listed for reference, all of these publications are ionized. Composition comprising a cationic lipid to be obtained and a liposome having a transmembrane pH gradient Use of such gradients to control bilayer intra- and extra-membrane distribution Therefore, it does not disclose the fusability of ionizable lipids.                                Summary of the Invention   The present invention is directed to (i) neutral bilayer-preferring lipids and primers. Rotonizable cationic head groups and unsaturated acyls Outermost layer lipid bilayer consisting of a fusion-promoting effective amount of ionizable lipids with chains And (ii) a region adjacent to the outermost lipid bilayer including the aqueous solution having the first pH. And a liposome having a composition. This composition Also contains an aqueous solution outside the liposomes having a second pH, the first pH Is the pK of the ionizable lipid in the outermost lipid bilayer_aSmaller, second PH Is the pK of an ionizable lipid in the outermost lipid bilayer._aBigger than Thus, there is a pH gradient across the outermost lipid bilayer, which also The lipid that can be converted is accumulated in the inner monolayer of the outermost lipid bilayer.   The liposomes can be unilamellar liposomes and unilamellar liposomes. The membrane is preferably a large unilamellar liposome . The liposome may be a multilamellar liposome, The somes contain solutes entrapped within their aqueous compartment, It is preferred that the solute concentration in each aqueous compartment of the posome be substantially equal. Yes. The internal aqueous solution in the liposome is preferably a buffered aqueous solution, more preferably More preferably, it is a buffered aqueous solution having a pH of about 4.0. A buffered aqueous solution with a pH of 4.0 It is preferably an enoate buffer.   The effective fusion promoting amount of the ionizable lipid is typically the outermost layer of the liposome. The concentration of the ionizable lipid in the lipid bilayer is about 1 mol% to about 20 mol%. In the outermost lipid bilayer membrane, preferably within this range. A preferred fusion-promoting effective amount of ionizable lipids in the outermost bilayer is Sufficient to bring the concentration of lipids that can be enantiomerized to about 5 mol% to about 10 mol%. .   The cationic headgroup of the ionizable lipid is preferably an amino group The unsaturated acyl chain is preferably an oleic acid chain. Presently Preferred Fruits of the Invention In an embodiment, the cation head group is the amino group and the unsaturated acyl chain is the ole group. In-acid chains and ionizable lipids are 1-N, N-dimethylaminodioleylpropayl. (AL-1). Ionizable lipids are ± -oleoyl-2-hydroxyl. Ci-3-N, N-dimethylaminopropane (AL-2), asymmetric ± -1,2-dia Syl-3-N, N-dimethylaminopropane (AL-3 to AL-5) and ± -1 , 2-didecanoyl-1-N, N-dimethylaminopropane (AL-6) You can also choose from a group. Currently, the ionizable lipid is more preferably It is 1-N, N-dimethylaminodioleoyl propane (AL-1).   The aqueous solution outside the liposomes is a solution of ionizable lipids in the outermost lipid bilayer. pK_aA buffered aqueous solution having a higher pH is preferred. Ionize When the obtained lipid is AL-1, the pH of the external buffered aqueous solution is about 7.5. Is preferred. Liposomes also include neutral, non-bilayer membrane-forming lipids, such as dipeptides. Oleoyl phosphatidyl ethanolamine (DOPE) can be included. The liposome may contain a physiologically active agent, typical examples of which include nucleic acid and bactericidal agent. , An anti-cancer agent or an anti-inflammatory agent. The aqueous solution outside the liposomes is pharmacologically acceptable. Aqueous solution, the liposome composition may be a pharmaceutical composition. it can.   The present invention is directed to neutral bilayer-forming lipids and cationic headgroups and satiety. Outermost Layer Lipid Consisting of a Fusion-Promoting Effective Amount of Ionizable Lipid Having Japanese Acyl Chain The ionizable lipid that has a molecular membrane is the outermost layer. It also provides dehydrated liposomes accumulated within the monolayer inside the lipid bilayer. It is also   Furthermore, it controls the fusion of liposomes to a second lipid bilayer. A method is provided. This method consists of preparing liposomes in an aqueous solution. The liposome comprises (1) a neutral bilayer-forming lipid and a protonatable positive ion. Fusing-promoting effective amounts of ionizable groups with on-head groups and unsaturated acyl chains Outermost lipid bilayer consisting of lipids, and (2) outermost lipid bilayer containing aqueous solution And a compartment adjacent to the membrane. The pH of the aqueous solution depends on the pH in the outermost lipid bilayer. PK of lipid that can be turned on_aLess than. Then, of the aqueous solution outside the liposome pH is the pK of an ionizable lipid in the outermost lipid bilayer_aHigher than This establishes a pH gradient across the outermost lipid bilayer. Can be ionized Lipids accumulate in the monolayer inside the outermost lipid bilayer, depending on the pH gradient. You. When the fusion of the liposome to the second lipid bilayer occurs, this pH gradient As a result, the liposome fuses with the second lipid bilayer.   The liposome used in the method of the present invention may contain a bioactive agent. , Representative examples thereof are nucleic acids, bactericides, anticancer agents or anti-inflammatory agents. Liposomes The second lipid bilayer that is fused in a controlled manner is the plasma membrane of the cell. Are preferred, and most preferably the plasma membrane of mammalian cells.   The present invention includes the agent in the aqueous solution such that the aqueous solution is inside and outside the liposome. A method for introducing a physiologically active agent into cells, including preparing liposomes. Offer. Liposomes consist of neutral bilayer-forming lipids as well as cationic head groups. And an Io having an unsaturated acyl chain It is composed of the outermost lipid bilayer including a lipid capable of being hydrolyzed. The pH of the internal aqueous solution is the outermost PK of ionizable lipids in layered lipid bilayers_aLower than. P of external aqueous medium H has a pH gradient across the outermost lipid bilayer and can then be ionized The outermost layer lipid is used so that the lipid is accumulated in the inner monolayer of the outermost lipid bilayer. PK of ionizable lipids in bilayers_aWill be higher than. PH of the internal aqueous solution Can ionize in the outermost lipid bilayer before cells contact the liposomes PK of lipid_aWill be higher than. The cells are preferably mammalian cells .                            Brief description of the drawings FIG.   (A) EPC / Chol liposomes containing 0-10 mol% AL-1 Calibration curve of TNS fluorescence. x-axis: AL-1 mol%; y-axis: fluorescence. Error bar (erro r bar) is smaller than the circle if not shown. (B) EPC / Cho pH gradient and 10 mol% AL- on TNS fluorescence of 1 (55:45) liposome Fluorescence trace showing effect of 1. Liposomes consisted of 0 mol% AL-1 (lower Race) or 10 mol% AL-1 (top trace). Inside Buffer is 300 mM citrate, pH 4.0 (lower trace) or Was 20 mM HEPES buffer, pH 7.5 (top trace). x Axis: time (seconds); y-axis: fluorescence.FIG.   Effect of pH on fluorescence of EPC / Chol (55:45) liposomes. Data shown represent mean values from duplicate experiments, error bars shown elsewhere. If not, it is smaller than the circle. The dashed line is the inverted first derivative of the titration curve. irst derivative) and its High values show a PK of 6.7 for AL-1 in lipid bilayers._aIs shown. x-axis: pH; y-axis:% ΔF / ΔF_maxIt is.Figure 3   EPC / Chol (55:45) Liposols with RET fluorescent probe dilution And fusion of EPC / DOPE / Chol (30:25:55) liposomes Effect of 0 and 10 mol% AL-1. The data shown is for the pH gradient to disappear Different traces of the experiment with and without_maxIs the By adding Triton X-100 to a final concentration of 0.8 mM, I asked. x-axis: time (seconds); y-axis: fluorescence.FIG.   EPC / Chol (55:45, mol / molar) diluted with RET fluorescent probe Le) Effect of AL-A concentration (0 to 20 mol%) on fusion of liposomes. x-axis: Time (seconds); y-axis: fluorescence.FIG.   EP in 100 mM ammonium acetate at pH 4.0 and 7.5 10 mol% AL-1-d in C / Chol (55:45)_FourOf the MLV prepared in₂ 1 H-NMR spectrum. x-axis: frequency (kHz).Figure 6   Effect of DOPE concentration on fusion rate of AL-1 containing liposomes. Fusion measurement The determination was performed as described above. x-axis: time (seconds); y-axis:% ΔF / ΔF_maxIn You.Figure 7   0 and 5 mol% AL-1 and EPC / Chol / DOPE (35:20 : 45, mol / mol). x-axis: time (seconds); y-axis:% ΔF / ΔF_maxIt is.Figure 8   20 mM HEPES, 20 mM sodium acetate at pH 4.0 and 7.5 Ium, buffered with 150 mM sodium chloride, EPC / DOPE / Chol 5 mol% AL-1-d in liposomes_FourKey Freeze-thaw MLVs made, A:₂H-NMR (x-axis: frequency (kHz)) spectrum Cutle, B:₃₁P-NMR (x-axis: δ / ppm).FIG.   EPC / Chol / DOPE (35:20 by dilution with RET fluorescent probe) : 45) Effect of aminolipids on fusion of liposomes. Trace (one from the top Bottom): AL-1, AL-5, AL-4, AL-3, AL-6, AL-2 and blank. x-axis: time (seconds); y-axis:% ΔF / ΔF_maxIt is.FIG.   Synthetic amino lipid structure.                            Detailed description of the invention   The present invention relates to (i) a neutral bilayer-forming lipid and a protonatable cation. A fusion promoting effective amount of ionizable fat having a head group and an unsaturated acyl chain Of the outermost lipid bilayer consisting of a substance, and (ii) an aqueous solution having a first pH (internal water Solution) and a liposome having a compartment adjacent to the outermost lipid bilayer containing A liposome composition is provided. The composition comprises liposomes having a second pH. It also contains an aqueous solution outside the. The first pH is the ion in the outermost lipid bilayer. Of lipidizable lipid_aThe smaller, second PH is the ion in the outermost lipid bilayer. Possible lipid pK_aGreater than, thereby crossing the outermost lipid bilayer There is a pH gradient, and depending on the pH gradient, the ionizable lipid is the outermost lipid bisecting. It is accumulated within the inner monolayer of the child membrane.   Liposomes are self-assembled structures consisting of one or more bilayer membranes, Each of them surrounds a compartment containing an aqueous solution. Each bilayer membrane of liposome Are polar, hydrophilic headgroups oriented towards the surrounding aqueous solution, while hydrophobic The reactive acyl chain toward the inside of the bilayer membrane A combination of amphipathic lipid molecules that orient themselves and move away from the surrounding aqueous phase. It is formed by setting. As a result, the lipid bilayer is separated into inner and outer monolayers of the lipid molecule. It has both child membranes.   Unilamellar liposomes have one lipid bilayer and The membrane has two or more lipid bilayers. Used in the liposome composition of the present invention The liposomes are unilamellar liposomes, preferably large unilamellar liposomes (L UV). LUVs have unit diameters greater than about 50 nm. It is a lamellar liposome. The liposome is a multilamellar liposome (MLV). MLVs may include solutes entrapped within the aqueous compartment and It is preferred that the solute concentrations in the fractions are substantially equal. Such an MLV is essentially Have an equal lamellar solute distribution. Generally, the liposome of the present invention is Izzes are about 5000 nm or less, as measured by their diameter. Liposome Size is well known to those of ordinary skill in the art and can be easily implemented by techniques such as It can be measured by quasi-electric light scattering.   The "outermost lipid bilayer" of unilamellar liposomes is a single liposome It is a lipid bilayer membrane of the The "membrane" is a lipid membrane that is in contact with an aqueous solution outside the liposome (the "external aqueous solution"). It is a molecular film. Liposomes can be prepared by a variety of methods (eg, Cullis et al. 1987), Bangham et al. (1965), Lenk et al. (US Pat. No. 4,522,8). No. 03, No. 5,030,453 and No. 5,169,637), Fountain Et al. (US Pat. No. 4,588,578) and Cullis et al. (US Pat. No. 4,975). , 282).   Liposomes are neutral (uncharged, non-cationic / non-anionic, aprotic) Includes a neutral bilayer-forming lipid consisting of a degroup and a bilayer-forming acyl chain It has an outermost lipid bilayer. These acyl chains have symmetry (length or carbon It may be asymmetric, with non-uniform child numbers, or may contain double bonds between adjacent carbon atoms. Not saturated, but containing one or more double bonds between adjacent carbon atoms ) It may be unsaturated and is generally the acyl of another lipid incorporated into the bilayer. Bilayer membranes by employing compatible acyl chains that are packed with the chains It is considered to suppress or prevent the phase separation of the non-bilayer membrane-forming lipid therein. Two minutes Child-forming lipids are related to other bilayer-forming lipids and non-bilayer-forming lipids. Can form a stable lipid bilayer on itself, as in . Bilayer-forming lipids generally consist of the surface area of their head group and their acyl chains. Has a substantial similarity with the cross-sectional area of The acyl chains of bilayer-forming lipids Generally, in a bilayer film, they are oriented substantially parallel to each other. Bilayer-forming lipid Generally have a bilayer-compatible structure and are generally involved in the production of bilayer defects. Not. Neutral bilayer-forming lipids are phosphatidylcholine-like It can be fatidylcholine or other neutral bilayer forming lipid.   The outermost lipid bilayer of the liposome is a cationic head group that can be protonated. That is, it accepts protons, is positively charged, and emits protons to become neutral. A fusion promoting effective amount of an ionizable lipid having a head group capable of Also included. Also, the ionizable lipid is an unsaturated acyl chain, preferably two. It also contains an unsaturated acyl chain. Cation head group is dimethylamino Group or trime It is preferably an amino group such as a tylamino group, but is protonated to give a positive Other groups that become charged and deprotonated to become neutral may be used.   Unsaturated acyl chains are generally non-bilayer forming. Non-bilayer forming Sil chains generally have a structure in which the chains are oriented parallel in the bilayer membrane. No. The cross-sectional area of such acyl chains is generally determined by the bilayer-forming acyl. Unlike the chains, they are not equal to each other nor to the head group surface area. this Is believed to be due to the non-parallel orientation. Non-bilayer forming acyl Bilayers containing chain-containing lipids generally form bilayers of the same length (number of carbon atoms). Compared with a bilayer membrane containing an acyl chain lipid, T_m, That is, the transition from gel to fluid state The temperature that occurs is lower. When a non-bilayer-forming lipid is incorporated into a bilayer, In general, defects are formed in the bilayer, making it less stable than using bilayer-forming lipids. The tendency to become. Such defects are commonly associated with lipid bilayer fusion. It is thought that Although not intended to be limited by theory, Destabilization, and therefore fusion, involves the size of the fusogenic lipid headgroup and the acyl chain. A substantial imbalance between the size of the area occupied by is considered necessary.   A preferred unsaturated acyl chain is an oleic acid chain, which has between the 9th and 10th carbon atoms. It is an acyl chain having 18 carbon chains with a double bond. However, unsaturated acyl chains As long as the lipids are firmly attached to the bilayer and are non-bilayer forming , Generally having 12 to 24 carbon atoms and 1 to 4 double bonds, for example palmy Trainate (16 carbon atoms, double bond 1) or arachidonate (20 carbon atoms) , Other double bond 4) and other acyl chains. Cation head group Is an amino group, the unsaturated acyl chain is an oleic acid chain, and can be ionized. It is preferred that the lipid containing two such chains comprises 1-N, N-dimethylamido. Nodioleoyl propane (AL-1) is preferred. AL-1 is optically active Or a racemate, that is, an optically inactive mixture of isomers, but a racemate Preferably.   Other suitable ionizable lipids are titratable head groups, ie, permanent It does not have a positive charge and is rather protonated or deprotonated in response to changes in ambient pH. A head group that is tonified and a lipid that has a non-bilayer-forming acyl chain It is quality. These include ± -oleoyl-2-hydroxy-3-N, N-di- Methylaminopropane (AL-2), asymmetric ± -1,2-diacyl-3-N, N- Dimethylaminopropane (AL-3 to AL-5) and ± -1,2-didecanoyl -1-N, N-dimethylaminopropane (AL-6) may be mentioned. It is not limited. The fusogenic capabilities of each of these lipids are described, for example, in Prepare liposomes with equal amounts of lipid each and then adjust the relative rate of liposome fusion. You can compare them by typing. Liposome fusion is a number of means, including For example, it can be monitored by a fluorescent probe dilution experiment (see, eg, Example 4 below). Can be. FIG. 9 shows ionizable lipids AL-1, AL-2, AL-3, AL Comparing the relative fusion rates of liposomes containing -4, AL-5 and AL-6. Data.   The liposome is a lipid in the outermost layer containing an aqueous solution (internal aqueous solution) having a first pH. It has a compartment adjacent to the molecular membrane. The liposome is an aqueous solution having a second pH (external Part aqueous solution). Typically, liposomes are prepared in aqueous solution, The aqueous solution is taken up by the liposomes, in which the liposomes It is suspended. Therefore, The inner and outer aqueous solutions generally initially have the same composition and same-pH. That Their pH depends on the ionizable lipid in the outermost lipid bilayer of the liposome. pK_aLess than.   PK of compound_aThat is, the acid dissociation constant is the pH at which the compound half dissociates, The pH is such that about half of the compound molecules present in the solution are deprotonated. pK_a Is the expression log ([HA] / [H₊] [A_-)) Can be defined here, And HA is a protonated compound, A_-Is a deprotonated compound. Henderson Hasselbach's formula (pH = pK_a+ Log ([A_-] / [HA] )) Is the pH of the solution and the protonated and depleted compounds present in the solution. It describes the relationship between the relative concentrations of protonated ones. In the lipid bilayer Its pK_aIonizable lipids present in bilayers at higher pH More than half of it is deprotonated and therefore neutral. Ionizable lipids PK_aIs a well-known and easily performed means, such as TNS fluorometric titration. Can be done by   The pH of both the internal and external aqueous solutions is their pK in the bilayer._aLower than When ionizable lipids are substantially protonated, they are generally the outermost lipid lipids. The monolayers on the inside and outside of the molecular film are distributed almost uniformly. That is, Io Approximately 50% of the lipid that can be enantiomerized exists in the inner monolayer and 50% in the outer monolayer. I do.   PK of the ionizable lipid in the bilayer above the first pH_aGreater than Raising the pH of the external aqueous solution to obtain an external aqueous solution with a threshold second pH Establishes a pH gradient across the outermost lipid bilayer. Is protonated, Charged, ionizable lipids, depending on the pH gradient, are inside the outermost lipid bilayer. Accumulates in the monolayer. As used herein, "accumulation" means that there is a pH gradient across the bilayer. In addition, about 50% or more of the ionizable lipids present in the outermost bilayer membrane are contained inside the monolayer. Being within the molecular membrane, preferably about 75 to about 100%, more preferably about 90 to about 100%, most preferably about 100% protonated, ionized It means that the lipid obtained is within the inner monolayer depending on the pH gradient.   For example, in a preferred embodiment of the invention, the ionizable lipid is AL- It is one. AL-1 with eg egg phosphatidylcholine (EPC) and cholesterol Roll (Chol) or EPC, Chol and Dioleoylphosphatidylco Liposomes can be prepared with phosphorus (DOPE). EPC, Chol And DOPE are neutral lipids, so EPC / Chol or EPC / Cho pK of AL-1 in 1 / DOPE bilayer_aIs about 6.7. Therefore, AL -1 / EPC / Chol or AL-1 / EPC / Chol / DOPE liposome , The first pH, ie the pH of the internal aqueous solution, is lower than 6.7, The first pH for AL-1 containing liposomes is preferably about 4.0. Generation Expressly, the internal aqueous solution is a buffered aqueous solution, and the preferred buffered aqueous solution is now a It is an enoic acid buffer.   The liposome is AL-1 / EPC / Chol or AL-1 / EPC / Chol / If DOPE is included, the second pH, ie, the pH of the external aqueous solution, is greater than 6.7. Preferably about 7.5. However, other lipids were When mixed with C / Chol or AL-1 / EPC / Chol / DOPE, there , AL-1 pK_aMay be affected. Ionizable other than AL-1 Lipid has different pK from AL-1_a May have. Therefore, the first and second p H If the composition of the liposome changes, you may change it from its preferred pH value. Yes.   PK of an ionizable lipid whose first pH is in the outermost lipid bilayer_aWhen lower , The second pH is its pK_aLarger, ionizable lipids are outermost lipid bimolecules The positive charge of lipids that can accumulate and ionize inside the monolayer inside the membrane is substantially outside. It is missing from the side monolayer and is shielded from exposure to the external environment. Movement When positively charged, positively charged lipids cause toxic side effects and lead to opsonization. Facilitates opsonization, ie, binding of plasma proteins to the outer surface of liposomes Can cause the clearance of liposomes from the circulation of the animal. ) Promote. Accumulation of a positive charge on the inner monolayer can cause toxic side effects and opsonin. Minimal potential for action.   Furthermore, the pH gradient that induces the accumulation of ionizable lipids on the inner monolayer is: Cationic lipophilic bioactive agents such as anthracycline antineoplastic agents, doxorubicin Can also be used to load liposomes (eg, Bally et al. See U.S. Pat. No. 5,077,056. ).   Due to the lowering of the pH gradient, the internal pH will be the pK of the ionizable lipid._aHigher than Become. This causes the accumulated ionizable lipids to be substantially deprotonated. Will be done. Then, a substantially deprotonated neutral ionizable fat. Quality is almost evenly distributed between the inner and outer monolayers of the outermost lipid bilayer . Neutral, ionizable lipids are exposed to other lipid bilayers within the outer monolayer. If it is, it is "fusional". That is, it promotes fusion of the liposome with other lipid bilayers. Can proceed. PK lower than about 7 in bilayer_aHaving an ion The use of derivatizable lipids ensures that the pH gradient is physiological, for example in animals. pH-decreasing, ionizable lipids are substantially deprotonated and fusogenic Means that. The internal pH of the liposomes depends on the substantial decapsulation of ionizable lipids. It is generally not necessary to raise it above physiological pH to induce rotonization. Substance Higher than physiological pH_aIonized lipids having , May not be fusogenic because it is not substantially deprotonated in animals .   The ionizable lipid is present in the outermost lipid bilayer in a “fusion promoting effective amount”. I do. The fusion of liposomes with other lipid bilayers is the outermost lipid bilayer of the liposome. It can be defined as the fusion of a membrane with other lipid bilayers, eg cell membranes (eg See, for example, Huang (1983)). For the fusion to occur, the outermost lipid bilayer Membrane lipids must mix with other lipid bilayer lipids. By theory There is no intention to be bound by this, but in order for fusion to occur, a The dead group must be neutralized, effectively changing the lipid morphology, and non-dichotomous. It is thought to give rise to a film-forming structure.   For purposes of this invention, a "fusion promoting effective amount" of an ionizable lipid is an ion. When the cationizable lipid is deprotonated and becomes neutral, another lipid It is the amount of ionizable lipid effective to promote fusion to the molecular membrane. Ionization A possible fusion-promoting effective amount of lipid will generally be such that a bilayer fuses with another bilayer. Is effective in creating defects in the bilayer that are sufficient to promote Typically, the “fusion promoting effective amount” of the ionizable lipid is the outermost lipid bilayer membrane. Sufficient to establish a concentration of ionizable lipid in the range of about 1 mol% to about 20 mol% Amount. Desirably, a fusion promoting effect of ionizable lipid The amount is about 5 mol% to about 10 mol% of the ionizable lipid in the outermost lipid bilayer. An amount sufficient to establish the concentration of.   Liposomes can also include neutral non-bilayer forming lipids. Here for If present, a “neutral non-bilayer-forming lipid” is a non-cationic, non-anionic head. It is an amphipathic lipid having a degroup and a non-bilayer-forming acyl chain. Non-division The child membrane-forming acyl chains are generally not arranged in parallel orientation in the bilayer membrane, In addition, the area generally occupied by the surface of the head group and the cross section of the acyl chain is Not the same. For such acyl chains, the most preferred packing structure is Generally, it has a non-bilayer structure. Bilayer membranes containing non-bilayer-forming lipids are generally In comparison with bilayers containing the same length (number of carbon atoms) of bilayer-forming acyl chain lipids. All T_mThat is, the temperature at which the transition from the gel to the fluidized state occurs is lowered. Therefore, Non-bilayer forming lipids generally facilitate the transition of bilayers from gel to fluid state , Thereby generally promoting the fusion of the bilayer to other bilayers. Departure In a presently preferred embodiment of Ming, the neutral, non-bilayer forming lipid is a Rail phosphatidyl ethanolamine (DOPE).   Liposomes are not known to those skilled in the Based on the amounts and reasons that can be determined by one skilled in the art, proteins and other lipids such as It is also possible to include sterols and their derivatives.   Liposomes are bioactive agents including conventional drugs and in vivo or in vivo animal cells. A related bioactive compound or composition having bioactivity in a cell can be included. Menstruation Active agents include antibacterial agents, antiviral agents, antifungal agents, antiparasitic agents, antitumor agents, and Xie Antagonist, Carbohydrate, Polypepti Peptide, protein, toxin, enzyme, hormone, neurotransmitter, glycoprotein, lipo Proteins, immunoglobulins, immune modulators, vasodilators, dyes, radiolabels Compounds, radiopaque compounds, fluorescent compounds, polysaccharides, cell receptor binding molecules, Anti-inflammatory agents, mydriatic compounds, local anesthetics, narcotics, anti-glaucoma agents, vitamins, nucleic acids, po Includes oligonucleotides, nucleosides, nucleotides, MRI and radiocontrast agents However, the present invention is not limited to these.   The liposome composition is administered to animals, preferably mammals, more preferably humans. Provided, incorporated into liposomes, or bound to liposomes The bioactive agent can be delivered to the cells of the animal. Where the composition is administered to animals If the external aqueous solution is tolerated, i.e., substantially non-toxic to animals, . Thus, an external aqueous solution is a pharmacologically acceptable solution, or "carrier." You. Generally, the pharmaceutically acceptable carrier is the intended administration route and standard. Selected based on common pharmacological practices. Intravenous, intraperitoneal, intramuscular, skin Liposomal composition for parenteral administration or injection via inferior or intramammary route A sterile solution may be prepared and the total concentration of solutes adjusted to render the product isotonic. Non-tradition A typical carrier used for oral administration is D5W (5% weight / volume Dextrose-containing aqueous solution (such as dextrose / water) and physiologically acceptable Salt solutions that may be used include, but are not limited to. Pharmacologically allowed Acceptable carriers include alcohols, gum arabic and benzyl alcohol. Carbohydrates such as starches, gelatin, lactose, amylose or starch, steari Magnesium acid salt, talc, silic acid, hydroxymethyl cellulose And polyvinylpyrrolidone. They have ingredients such as preservatives, antioxidants, etc. within the range that can be determined by those skilled in the art. It can be included based on quantity and reason.   The present invention is directed to neutral, non-bilayer forming lipids as well as protonatable cationic heads. Is a Fusion-Promoting Effective Amount of Ionizable Lipids Having a Dogroup and Unsaturated Acyl Chain? It is a dehydrated liposome having an outermost lipid bilayer consisting of However, the dehydrated liposomes accumulated in the monolayer inside the outermost lipid bilayer are also included. provide. Dehydration of liposomes can extend the shelf life of liposomes. The dehydrated liposomes can be reconstituted if necessary. Liposomes Can be dehydrated by freezing using a standard freeze-drying device or its equivalent You. Freeze-drying is described by Schneider et al. (US Pat. , 360) and Janoff et al. (US Pat. No. 4,880,635). According to the method, after mixing one or more protective sugars into the liposome product, Is preferred. Protective sugars should be omitted if dehydration is carried out without freezing. And leave sufficient water in the liposome product during the liposome preparation process. Results in the completion of a substantial portion of the liposome bilayer membrane during the dehydration-rehydration process. The integrity can be maintained.   The present invention comprises preparing liposomes in an aqueous solution, the liposomes comprising Neutral bilayer-forming lipids and protonatable cation headgroups and Outermost layer lipid containing a fusion-promoting effective amount of an ionizable lipid having an unsaturated acyl chain Proposed a method to control the fusion of liposomes to a second lipid bilayer, including the bilayer. Offer. Liposomes also contain a compartment adjacent to the outermost lipid bilayer that contains an aqueous solution. It is. The pH of the aqueous solution depends on the ionizable lipid in the outermost lipid bilayer. p K_aLower than. Then, the pH of the aqueous solution outside the liposome is adjusted to the outermost lipid bimolecule. PK of ionizable lipids in membranes_aSince it is higher than the A pH gradient is created across which the ionizable lipids are the outermost lipids depending on the pH gradient. Accumulates on the inner monolayer of the bilayer. Liposome fused with a second lipid bilayer If the pH of the inner aqueous medium is adjusted to that of the ionizable lipid in the outermost lipid bilayer. pK_aHigher than. The liposomes are unilamellar liposomes, preferably large liposomes. Nilamella liposomes, or multilamellar liposomes, preferably substantially etc. It can be a multilamellar liposome with a new interlamellar solute distribution. second The lipid bilayer of is preferably a plasma membrane of a cell, and the cell is a mammalian cell. preferable. However, the second lipid bilayer is a liposome lipid bilayer or It may be another lipid bilayer membrane such as a cell membrane of bacteria. The pH of the external aqueous solution is In general, does the aqueous solution contain enough base to obtain the desired pH? , Or its external aqueous solution is replaced with a second aqueous solution having a desired pH. To make it higher.   The ionizable lipid is a fusion promoting effective amount, that is, the second lipid bimolecule of the liposome. It is present in the outermost lipid bilayer in an amount effective to promote fusion to the membrane. Generation Tableically, the “fusion-promoting effective amount” of the ionizable lipid is in the outermost lipid bilayer. At about 1 mol% to about 20 mol% of ionizable lipid concentration is established. Is enough. Preferably, the fusion promoting effective amount of ionizable lipid is the outermost In the layered lipid bilayer, from about 5 mol% to about 10 mol% of the ionizable lipid. A sufficient amount to establish the concentration.   If you want to fuse liposomes to a second lipid bilayer, pH gradient Internal pH of the liposome can be ionized in the bilayer so as to reduce PK of lipid_aHigher than. Accumulated in the inner monolayer of the outermost lipid bilayer Entrapped, protonated / charged, ionizable lipids are PH of aqueous solution is lipid pK in bilayer membrane_aHigher than Be protonated. It may then be substantially deprotonated / neutralized The lipid is distributed almost uniformly in the outermost lipid bilayer. That is, about 50% is inside In the monolayer, about 50% is present in the outer monolayer. The neutral lipid in the outer monolayer is , Can promote fusion to other lipid bilayers. Act on the liposome composition As the protons stored in the liposomes leak to the external environment, It is possible to reduce the pH gradient by gradually eliminating it in the animal. it can. As the internal pH of liposomes approaches physiological pH, physiological p PK lower than H_aIonizable lipids with are substantially deprotonated . Ionophores that facilitate the transport of ions across lipid bilayers, such as Nigeri Lipid dichotomization, either by using nigericin, or in a neutral form Adding an ion that can cross the child membrane, such as an ammonium ion Therefore, the pH gradient can be reduced. Internal pH is pK_aHigher than The charged, ionizable lipids are then substantially deprotonated to give neutral lipids. Are distributed almost uniformly between the inner and outer monolayers of the outermost lipid bilayer.   The liposome used in the method of the present invention can contain a bioactive agent. . The second lipid bilayer that controls the fusion of lipids is preferably the plasma membrane of the cell, The cells are preferably mammalian cells. In mammals, ie in vivo, Membrane to the plasma membrane of mammalian cells Preferably. Typically, fusions are those in which the liposome content is of mammalian cells. It occurs as it is transported to the cell membrane.   It also comprises preparing liposomes containing a physiologically active agent in an aqueous solution. Posomes are neutral bilayer-forming lipids as well as a protonatable cation head. Containing a fusion promoting effective amount of ionizable lipids having groups and unsaturated acyl chains. A bioactive agent containing an outermost lipid bilayer is introduced into cells, preferably mammalian cells. How to enter is also provided here. The aqueous solution contains the ions in the outermost lipid bilayer. Of lipidizable lipid_aHas a lower pH and is taken up by the formed liposomes And enclose the liposome. The liposome is the outermost layer containing an aqueous solution It also contains a compartment adjacent to the lipid bilayer. Then, the aqueous solution outside the liposome Of the pH of the ionizable lipid in the outermost lipid bilayer_aHigher than Therefore, a pH gradient is created across the outermost lipid bilayer and the ionizable lipid is Accumulates in the inner monolayer of the outermost lipid bilayer depending on the pH gradient of. Then For example, by administering liposomes to animals, or by In the case of fusion, the use of ionophores reduces the pH gradient. next , So that the liposome fuses with the cell, thereby introducing the bioactive agent into the cell First, the liposomes come into contact with the cells.   The invention will be better understood by the following examples. However, These examples simply illustrate the invention as defined in the claims which follow. It is easily understood by those skilled in the art that it is only explained.                                   Example Example 1 Lipid and chemical materials   Egg phosphatidylcholine (EPC), dioleoylphosphatidylethanol Amine (DOPE), N- (7-nitro-2,1,3-benzoxydiazole- 4-yl) -1,2-dioleoyl-sn-phosphatidylethanolamine ( NBD-PE) and N- (lissamine rhodamine B sulfonyl) ) -1,2-sn-phosphatidylethanolamine (Rh-Pe) is Avanti  Obtained from Polar Lipids (Alabaster, AL). Oleic acid, cholesterol ( Chol), nigericin, 2-p-toluidinylnaphthalene-6-sulfonic acid Lithium (TNS) and all buffers are from Sigma Chemical Co. (St. Louis, MO ). 3-N, N-dimethylamino-1,2-propanediol and Oxalyl chloride is commercially available from Aldrich Chemical Co. Purchased from (Milwaukee, WI), 10-d2-oleic acid was supplied by MSD Isotopes (Montreal, PQ). Existence All organic solvents were HPLC grade and were used without double distillation.Synthesis of AL-1   This compound was prepared by the method of Levantis and Silvius (1990). To 1.0 g (3.5 mmol) of oleic acid dissolved in 10 ml of benzene, ml (35 mmol) of oxalyl chloride was stirred at room temperature for 1 hour without stirring. The oleyl chloride was prepared by adding the oleyl chloride. Solvent and excess salt under vacuum After removing the oxalyl chloride, the acid chloride was dissolved in 5 ml of diethyl ether. , 0.20 g (1.7 mmol) of 3, N-dimethylamino-1,2-propyl 5 ml of ether containing lopandiol and 0.15 g of pyridine was added. The resulting mixture was stirred at room temperature for 30 minutes and then cooled with 1 ml of methanol. . The solvent was removed under vacuum. The crude product is dissolved in 50 ml of hexane, 2 x 25 Wash with ml 0.1 M sodium chloride. Dry over anhydrous sodium sulfate and vacuum The hexane was removed underneath to give a slightly yellowish oil. silica Column chromatography on gel (70-230 mesh) with ethyl acetate. Elution, 0.92 g (84%) of pure product (TLC, Rf = 0.5) was gotten. 200MHz, 1H-NMR (CDCl_Three), TNS (J Capri Δppm from integration (J-coupling, integration): 5 . 3 (dd, 4H), 5.1 (m, 1H), 4.0 (dd, 1h), 2.4 (d d, 2H), 2.2 (s, 6H), 2.0-0.8 (m, 62H). This way , A deuterated analog, rac-1-N, N- Dimethylamino-2,3-bis (9,10-dideuteriooleoyl) propa (AL-1-d_Four) And ± -1,2-didecanoyl-1-N, N-dimethylacetate It was also used to prepare minopropane (AL-6).± -oleoyl-2-hydroxy-3-N, N-dimethylaminopropane (AL -2) and asymmetric ± -1,2-diacyl-3-N, N-dimethylaminopropane ( Preparation of AL-3 to AL-5)   The oleoyl chloride (3.4 mmol) prepared as described above was added to 5 ml of T 5-fold excess of 3-N, N-dimethylacetate dissolved in HF and 25 ml of THF. Mino-1,2-propanediol (2.0 g, 17 mmol) and 0.15 g Added to pyridine. Crude 1-monooleoyl-2-hydroxy-3-N, N-di Methylaminopropane (AL-2) was isolated, and acetic acid was used as an eluent (Rf0.4). Ethyl / me By column chromatography on silica gel using tanol (3: 1). Refined. Then 1 equivalent of acetyl chloride, butyryl chloride or decanoyl chloride By acylation under the reaction conditions to produce AL-3, AL-4 and AL-5, respectively. Was manufactured.Preparation of LUV   Dry the chloroform solution of the lipids under nitrogen, then continue under high vacuum to remove residual solvent. By removing the liposomes for 1 hour by a known method. mal liposome) was prepared. Swirl the resulting lipid film with an appropriate buffer ( vortex) to hydrate and produce multilamellar liposomes (MLV) I made it. Freezing and thawing was performed 5 times to obtain a uniform mixture. MLV with two pore supports Extrude 10 times from a polycarbonate filter with 100 nm, Lamellar liposomes have been prepared (eg, Bally et al., US Pat. No. 4,885,17). No. 2, Cullis et al., U.S. Pat. No. 4,975,282, Cullis et al., U.S. Pat. No. 5, , 008,050, Loughrey et al., U.S. Pat. No. 5,059,421).Example 2 Measurement of lipid asymmetry by TNF fluorescence   Transport of AL-1 to the inner monolayer of liposomes was described by Eastman et al. (1991). Was demonstrated by TNF fluorescence using the method adopted from First, EPC / Cho 1 (55:45), 0, 2.5, 5.0, 7.5 and 10.0 mol% AL-. 1 in 20 mM HEPES, 150 mM NaCl, pH 7.5, total lipids TNF fluorescence as a function of AL-1 concentration was assayed using 5 mM LUV. Part of 90 μl 20 mM MHEPES, 150 mM NaCl, 5 μM TNS, pH 7.5. Add to 2.9 ml and monitor fluorescence for 5 minutes. As mentioned above For comparison, liposomes prepared with 10 mol% AL-1 and an internal pH of 7.5 were also prepared. used.   The results are shown in Fig. 1. After a pH gradient was applied, TNF fluorescence was used to detect the AL-1-containing solution. Monitoring the surface charge of posomes and affecting the distribution of AL-1 across bilayers The effect of pH gradient was demonstrated. Surface charge increased at a concentration of 0.5 mol% AL-1 It was observed that the fluorescence increased uniformly with increasing temperature (see FIG. 1A). However At high concentrations, fluorescence levels off and begins to decline. This effect affects cholesterol It was not observed in the liposome containing AL-1 in EPC containing no fluorescence, and the fluorescence was 20 Increased linearly up to mol% AL-1. In EPC / Chol (55:45) Liposomes containing 10 mol% AL-1 aggregated and re-assembled for TNS interactions. Early signs of reduced posome surface area appeared.   When EPC / Chol (55:45) contains 10 mol% AL-1, The effect of pH gradient on TNF fluorescence is illustrated in Figure 1B. The curve at the top of the figure is For 10 mol% AL-1, there was no gradient inside and outside at pH 7.5. The fluorescence is shown as a function of time. The bottom curve is for vesicles, It has an internal pH of 4 and an external pH of 7.5, but does not contain AL-1. these The slow decrease in fluorescence observed in the curve of TNS is due to the photobleaching (phot obleaching) is the result. The remaining curves are 10 mol% AL- with a slope. 1 for most of the increased surface charge of the liposome, thus AL- 1 was lost very rapidly from the outer monolayer. Is shown. After 4 minutes, the fluorescence was equal to that of EPC / Chol liposomes and Qualitatively, it was shown that all AL-1 had translocated to the inner monolayer. two The lipid translocation of AL-1 across the molecular membrane is useful for its use in controlling fusion. is there. Similar behavior was also observed for AL-2 to AL-6 (see FIG. 9).Example 3 Measurement of pK _a of lipids that may be ionized   To measure the pH at which AL-1 in the liposome membrane loses its charge, LUV containing 0 and 10 mol% AL-1 and EPC / Chol (55:45), Prepared at pH 4.0 in 5 mM HEPES, 5 mM ammonium acetate. 5 3.0 to 1 in mM HEPES, 5 mM ammonium acetate, 2 μM TNS Samples were diluted to 0.25 mM total lipid in a pH range up to 0.0 . Vesicles were added by adding nigericin to a final concentration of 0.01 μM. The pH across the membrane was equalized.   The results are presented in Figure 2 and show various pH values in the absence of a pH gradient. The TNS fluorescence in the values is shown. From this data, for the conjugate acid of AL-1, pK_aIs clearly 6.7. Synthetic amino lipids AL-2 to AL-6 Table 1 shows a comparison of the results derived from similar pH titration curves (see below). ). When incorporated into liposomes, the effect of chemical structure on the acid-base properties of amines The effect is the observed pK_aAmino fat that remains unchanged at pH value and pH 7.5 It is expressed as a change in the fraction of a species. These changes depend on the relative hydrocarbon chain length and Amine as it is affected by the polarity of the headgroup It can be derived from the depth of penetration of the headgroup into the lipid bilayer. Compound The product AL-2 has only a single oleoyl chain and an unsubstituted hydroxyl group. Yes, pK_aIs about 7.57. Acetate the 2-position hydroxyl with acetate When converted to tellurium, it becomes AL-3, the pKa is lowered to 6.79, and at pH 7.5. The fraction of neutral species that is present is 16% compared to 11% for AL-1. Remaining compound Things AL-4, AL-5, AL-6 are also pK_aAnd the relative charge at PH 7.5 is , AL-1 with sufficient hydrocarbon content to approach those values. Example 4 Lipid-mixed fusion measurement   Diluted with a fluorescent probe, as described by Struck et al. (1981). Fusion was monitored by the resulting reduction in resonance energy transfer. pH 4.0 EPC / Chol (55:45) and EPC / D in 300 mM citrate With 0 and 10 mol% AL-1 in OPE / Chol (30:25:45) LUV was prepared. The external buffer is 20 mM HEPES (150 mM Na Cl, pH 7.5). For each lipid composition at each AL-1 concentration , Liposomes containing 0.7 mol% of both NDB-PE and Rh-PE were also prepared. Was. Labeled liposomes and unlabeled liposomes were mixed at a ratio of 1: 3, and The lipid was diluted to 0.2 mM. Sephadex G-25 On the column, add external buffer to 300 mM sucrose, 1 mM citrate, pH 4.0. The sample was then diluted to 10 mM lipid. 20μ for fusion measurement l fluorescence-labeled liposomes and 60 μl unlabeled liposomes were added to 3.9 Added to 2 ml of 20 mM HEPES, 150 mM NaCl, pH 7.5. After incubating for 5 minutes at room temperature, 3 ml was added to the cuvette for 5 minutes I monitored the fluorescence. Excitation and emission wavelengths are 465 nm and 53, respectively. 5 nm and an emission 530 nm cut-off filter was used. The gradient disappears In order to generate the fusion, acetic acid was added in 30 seconds to a final concentration of 100 mM. Added ammonium. Zero fluorescence (F₀) Is added without ammonium acetate. In the race Fit, F_maxWas measured after adding 100 μl of 25 mM Triton X-100. By fitting the traces, the percent change in fluorescence (% F / F_max) I asked. That is,   % (F / F_max) = 100 × ((F−F₀) / (F_max-F₀))   In order to examine the effect of changing the AL-1 concentration (0 to 20 mol%) on the fusion, Those with and without 0.5 mol% NBD-PE and Rh-PE, respectively For internal, pH 4.0, 300 mM sodium citrate buffer and external , 20 mM HEPES buffer (150 mM NaCl, pH 7.5) , EPC / Chol (55:45) containing liposomes were prepared. Labeled lipo The liposomes and unlabeled liposomes were mixed at a ratio of 1: 3, and the total lipid was 0.20 m. Diluted to M. Ammonium acetate so that the final concentration is 100 mM The pH gradient disappeared by addition of 30 seconds.   To study the effect of changing the DOPE concentration on the fusion rate of AL-1-containing liposomes For the purpose of EPC / DOPE / Chol (55-X: X: 45, X = 0, 5, 1, 0, 15, 20), 10 mol% AL-1 and 0.5 mol% NBD each -With and without PE and Rh-PE, an internal pH of 4.0 , 300 mM sodium citrate buffer and external, 20 mM HEPES buffer LUV with (150 mM NaCl, pH 7.5) was prepared.   Effect on fusion of EPC / DOPE / Chol (35:20:45) liposomes To study the effect of aminolipid structure, 5 mol% of the above aminolipids (AL-1 ~ AL-6), internal, pH 4.0, 300 mM sodium citrate buffer And external 20 mM HEPES buffer (150 mM NaCl, pH 7.5) And liposomes Was prepared. Fusion measurements were performed as described above.   The results are shown in Figures 3, 4, 6, 7 and 9. Liposomal membrane with acidic internal liquid The pH gradient across the ionophore is due to ionophores such as nigericin or to neutral ammonia. Ammonium ions capable of increasing internal pH across the membrane in the form of Ni Can be eliminated by adding. AL-1 exclusively inside Regarding LUV existing in the molecular film, AL-1 is a single molecule due to the disappearance of the pH gradient. It would be expected to be redistributed between the membranes. AL-1 is deprotonated to give two molecules Under conditions of membrane instability, liposome fusion can occur.   Membrane fusion is due to the disappearance of RET between the fluorescently labeled lipids NDB-PE and Rh-PE. I monitored it. Liposomes containing both of these labels are When fused, the resulting dilution of fluorescent probe is fluorescent for NDB-PE. Indicates an increase. The marked exchange between these labeled lipids between liposomes is due to the aggregation system. It does not seem to occur, even when fluorescence increases only when the membrane lipids are mixed. I do.   Fluorescent probe to demonstrate fusion in LUVs containing 10 mol% AL-1 The use of dilution measurements is shown in Figures 3 and 4. Including EPC / Chol (55:45) , Unlabeled liposomes without AL-1 and labeled liposomes (3: 1) disappeared pH gradient It does not show an increase in fluorescence. Due to the addition of ammonium solution, A decrease in light was observed. Similar liposomes containing 10 and 20 mol% AL-1 Then, ΔF / ΔF around 3%_maxAt values of, the fluorescence increases sharply. this is , Prepare a liposome with a fluorescent label at 1/4 of the normal concentration (0.18 mol%). As a result of the production, it was found that a 3: 1 mixture of unlabeled and labeled liposomes was obtained. Then 80% of ΔF / ΔF_maxShould give A limited amount of the total poss ible lipid mixing). The low fluorescence increase observed is A L-1 causes pH gradient-controlled fusion in EPC / Chol liposomes Can be rubbed but shows that its ability to do so is limited . For AL-1 concentrations greater than about 20%, EPC / Chol liposomes In, the abrupt and complete aggregation was observed after extrusion.   Dioleoylphosphatidylethanolamine (DOPE) on lipid bilayer By including it, the fusion property of the EPC / Chol liposome can be enhanced. FIG. 3 shows the effect of EPC / DOPE / Chol (35:20:45) fusion. 3 shows a fluorescence trace demonstrating the effect of mol% AL-1. Vinegar without amino lipids There was very little evidence of fusion when the pH gradient disappeared with ammonium acidate. I However, when AL-1 was included, ΔF / ΔF was reached in 5 minutes._maxIncreased to about 10% , Continued to rise for 15 minutes. This result shows that AL-1 in control fusion Demonstrate utility and explain importance of lipid composition in the extent of fusion observed Is what you do. Increased the concentration of DOPE instead of EPC, 10 mol The results of fusion measurement for EPC / DOPE / Chol containing% AL-1 , As shown in FIG. The lipid ratio of DOPE concentration is 35:20:45 (EPC / DOPE / Chole, mol / mol), the fusion rate increases it is obvious. This composition shows ΔF / ΔF after 5 minutes._maxIs greater than 5% . However, at this concentration of DOPE, at low pH there was some aggregation in the liposomes. Was recognized. When the DOPE concentration became high, agglomeration occurred rapidly.   In EPC / DOPE / Chol (35:20:45) liposomes, AL The best fusion rate observed when the concentration of -1 was reduced to about 5 mol% is shown in FIG. Show. With this composition, at pH 4.0, stable liposomes are prepared and Remained stable when was raised to 7.5. When the pH gradient disappears, the first ΔF / ΔF in 5 minutes_maxRises almost linearly above 10%. Fusion The coalescence rate decreased with time, but the fusion continued during the 15 minute measurement time.   Each amino lipid in EPC / DOPE / Chol (35:20:45) liposome By measuring the fusion of liposomes containing 5 mol% of The fusion ability of AL-1 to AL-6 was compared. FIG. 9 shows the results. Single oleoi AL-2, which has a poly-chain, is more liposomal compared to liposomes without aminolipids. Fume fusion does not increase much. The lack of fusion activity is associated with such bilayers. And the pK of AL-2_aIs relatively high and is occupied by a single acyl chain of AL-2. It is considered that this is due to the relatively small area in the molecular film. By theory The destabilization of the bilayer membrane, that is, fusion, is not limited by the fusogenicity. The substance between the size of the lipid head group and the size of the area occupied by its acyl chains. Imbalance is considered necessary. A liposome containing AL-3 is substantially It shows a greater fusion rate and lengthens the second strand of AL-3 to give butyryl (AL- If 4) or decanoyl (AL-5) is used, the fusion speed will be further increased, and AL- Approaching the rates achieved with 1-containing liposomes. Compounds with two decanoyl chains When the substance AL-6 is mixed with the liposome, the pK becomes relatively high._aLow but limited Only mixed mixing is done.Example 5 ² H-NMR spectroscopy   Using deuterium-free water, at pH 4.0 and pH 7.5, 100 mM acetate As described above, 10 mol of EPC / Chol (55:45) % AL-1-d_FourFreezing and thawing MLV containing was prepared. The sample concentration is It was about 150 mM. 5.3μs 90 ° pulse and 200ms repetition delay ( repeat echo) spin echo sequence at 30.7MHz Broad quadrupole spectra were recorded on a Bruker MSL200 spectrometer. Temperature is liquid The temperature was kept at 20 ° C. with an elementary flow system. Free-in for pH 4.0 samples A free induction decay (FID) signal is output overnight (about 28 50,000 scans), while the pH 7.0 sample is comparable. Signal-to-noise was performed in about 770 scans. 100Hz wide line The FID was converted using line-broadening.Example 6 ² H-NMR and ₃₁ P-NMR spectroscopy   20 mM HEP at pH 4.0 and pH 7.5 using deuterium-free water ES, 20 mM ammonium acetate, 150 mM NaCl with EPC / Chol ( 55:45) with 5 mol% AL-1-d._FourFreezing and thawing MLV containing was prepared. Sun The pull concentration was about 200 mM total lipid. 5μs 90 ° pulse, 50μs pulse Loss interval, 30.5 μs ring-down delay and 300 ms repetition With a homemade 300 MHz spectrometer at 46.175 MHz ,₂H -NMR broad spectra were recorded. Quadrupole echo system with 8-step phase cycling 200,000 scans were accumulated using Quens. Free inn obtained The induction decay (FID) was converted with a 100 Hz line spread. 2.8 μs Bruker MSL200 Spectrometer at 81 MHz using Ruth and 30s repetitions With high resolution₃₁The P spectrum was recorded. The temperature is 23 ° C with a liquid nitrogen flow system. Held in. Accumulate FIDs over 1000 scans and spread 50Hz line Converted in.   The results of NMR spectroscopy are shown in Figures 5 and 8. EPC / Chol (55:45) Liposo It was confirmed that the degree of fusion was limited for 10 mol% AL-1 in This is because the deprotonated form of aminolipids is not stable in bilayers. Although not, this instability did not give the lipid membrane a permanent non-bilayer structure. That was shown. This limits the solubility of neutral amines in the bilayer. It could have been the result of that. AL before widespread melting can occur -1 discrete crystalline or fluid regions may be formed.   Deuterium chain labeled analog, AL-1-d_FourOf 10 mol% EPC / Chol The phase behavior of AL-1 was adjusted by mixing with MLV at a concentration of (55:45). Solid. When using MLVs in this experiment, the LU Avoiding sudden V and shake. In FIG. 4, at pH 4.0²H-NMR The spectrum shows AL-1-d of the magnitude expected for lipids in bilayers.^Four The quadrupole splitting corresponding to the strand labeling position of is shown. At pH 7.5 , The bilayer signal is no longer present and there is a strong isotropic peak in the middle of the spectrum. Appears. This is a separate fluid region at pH 7.5. In addition, there is strong evidence that AL-1 is present. Neutralization and subdivision of AL-1 The limited degree of fusion observed in distribution is a result of the formation of these regions. Conceivable.   PH 4.0 in FIG. 8A²H spectrum was incorporated into the lipid bilayer Shows quadrupole deuterium splitting to the extent expected for lipid oleoyl chains . Quadruplet splitting was reduced in samples prepared at pH 7.5, This seems to be related to the lowering of the level of the bilayer membrane (order), , The bilayer remains. In addition, about 12kHz splitting is explained. There is a definite signal, which is H₁₁Generated from protons of aminolipids in phase It is considered to be. Thus, the appearance of this signal results in membrane fusion. Consistent with destabilization of the bilayer.   The attendant changes in the phase behavior of the phospholipids EPC and DOPE are shown in Figure 8B.³¹ It is apparent in the P-NMR spectrum. For samples with pH 4.0, top ( A typical two with a peak in the upfield and a shoulder in the bottom. A signal of the molecular film was observed. For the pH 7.5 sample, the bottom signal Is slightly reduced, and as a stronger lower shoulder, H₁₁You can see the signal.Example 7 Freeze-fracture microscopy   EPC / Chol (55:45) or EPC / DOPE / Chol (30: 2) 5:45), LUV consisting of 10 mol% in Prepared with ammonium enoate to a total lipid concentration of 150 mM as described above did. Sephadex G-25 Color Buffer to 1 mM citrate, 300 mM sucrose, pH 4.0. Changed. To 200 μl of each sample, 50 μl of 500 mM ammonium acetate, pH 7 . 5 was added. Hold the sample at room temperature for 30 minutes, then platinum / carbon replica Was prepared (see Fisher and Branton (1974)).Example 8 Freeze-fracture microscopy   LUV consisting of 5 mol% in EPC / DOPE / Chol (30:25:45) As described above with 300 mM ammonium citrate at pH 4.0. The total lipid concentration was adjusted to 100 mM. Sephadex G-25 column Replace the external buffer with 1 mM citrate, 300 mM sucrose, pH 4.0 Then, 20 mM HEPES, 150 mM NaCl so that the lipid becomes about 10 mM. The sample was diluted at pH 7.5. After removing the control sample, the final concentration was 10 3 mM ammonium acetate, pH 7.5 was added to adjust the pH gradient to 0 mM. Let it disappear. In addition to the control sample in which a pH gradient is present, 4, 15 and A platinum / carbon replica was prepared for the 30 minute sample (Fisher and And Branton (1974)).

────────────────────────────────────────────────── ─── Continuation of front page    (72) Inventor Chris, Peter, Earl             British Columbia, Canada             VI6 Earl 1 Etch 4 Van Cuba             -West First Avenue               3732

Claims (1)

[Claims] 1. (A) (i) an outermost lipid bilayer comprising a neutral bilayer-forming lipid and a fusion-promoting effective amount of an ionizable lipid having a protonatable cation head group and an unsaturated acyl chain; (Ii) a liposome having a compartment adjacent to the outermost lipid bilayer containing an aqueous solution having a first PH, and (b) an aqueous solution outside the liposome having a second PH, is smaller than the pK _a of lipids capable of ionizing in the outermost layer lipid bilayer, the second PH is greater than _the pK _a of lipids capable of ions in the outermost lipid bilayer, whereby the outermost lipid double A liposome composition, characterized in that there is a pH gradient across the molecular membrane, whereby ionizable lipids accumulate within the inner monolayer of the outermost lipid bilayer. 2. The liposome composition according to claim 1, wherein the liposome is a unilamellar liposome. 3. The liposome composition according to claim 2, wherein the unilamellar liposome is a large unilamellar liposome. 4. The liposome composition according to claim 1, wherein the liposome is a multilamellar liposome. 5. The liposome composition according to claim 4, wherein the multilamellar liposome comprises a solute incorporated in its aqueous compartment, and the concentration of the solute contained in each aqueous compartment of the multilamellar liposome is substantially equal. 6. The liposome composition according to claim 1, wherein the aqueous solution having the first PH is a buffered aqueous solution. 7. The liposome composition according to claim 6, wherein the pH of the buffered aqueous solution is 4.0. 8. The liposome composition according to claim 7, wherein the aqueous buffer solution is a citrate buffer solution. 9. The liposome composition according to claim 1, wherein the effective amount for promoting fusion of the ionizable lipid is an amount sufficient to bring the concentration of the ionizable lipid in the outermost lipid bilayer to 1 mol% to 20 mol%. . 10. 10. The liposome composition according to claim 10, wherein the effective amount for promoting fusion of the ionizable lipid is an amount sufficient to bring the concentration of the ionizable lipid in the outermost bilayer membrane to 5 mol% to 10 mol%. 11. The liposome composition according to claim 1, wherein the unsaturated acyl chain is an oleic acid chain. 12. The liposome composition according to claim 1, wherein the cation head group is an amino group. 13. Ionizable lipids are 1-N, N-dimethylaminodioleylpropane (AL-1), ± -oleoyl-2-hydroxy-3-N, N-dimethylaminopropane (AL-2), asymmetric ± Consists of -1,2-diacyl-3-N, N-dimethylaminopropane (AL-3 to AL-5) and ± -1,2-didecanoyl-1-N, N-dimethylaminopropane (AL-6). The liposome composition according to claim 12, which is selected from the group. 14． The liposome composition according to claim 13, wherein the ionizable lipid is 1-N, N-dimethylaminodioleoylpropane (AL-1). 15. The liposome composition according to claim 1, wherein the liposome further comprises a neutral non-bilayer-forming lipid. 16. The liposome composition according to claim 15, wherein the neutral non-bilayer forming lipid is dioleoylphosphatidylethanolamine. 17． The liposome composition according to claim 1, wherein the liposome contains a bioactive agent. 18. The liposome composition according to claim 17, wherein the physiologically active agent is a nucleic acid, a bactericidal agent, an anticancer agent or an antiinflammatory agent. 19. The liposome composition according to claim 1, wherein the outer aqueous solution is a pharmacologically acceptable aqueous solution. 20. An ionizable lipid having a neutral bilayer-forming lipid and an outermost lipid bilayer consisting of a protonatable cation head group and a fusion-promoting effective amount of an ionizable lipid having an unsaturated acyl chain is , A dehydrated liposome accumulated in a monolayer inside the outermost lipid bilayer. 21. (A) preparing a liposome represented by (i) (ii) in an aqueous solution, (i) said liposome is (1) a neutral bilayer-forming lipid and a protonatable cation head group and unsaturated An outermost lipid bilayer comprising an fusion-promoting effective amount of an ionizable lipid having an acyl chain, and (2) a compartment adjacent to the outermost lipid bilayer containing an aqueous solution, (ii) The pH of the aqueous solution is lower than the pK _a of the ionizable lipid in the outermost lipid bilayer, and (b) the pH of the aqueous medium outside the liposome is the ionizable lipid in the outermost lipid bilayer. the pK higher than _a, thereby, there is a pH gradient across the outermost lipid bilayer and whereby the lipid capable of ionization are accumulated in the inner monolayer of the outermost lipid bilayer, And (C) a second lipid bilayer comprising lowering the pH gradient when the fusion of the liposome to the second lipid bilayer occurs, such that the liposome fuses to the second lipid bilayer. Methods of controlling the fusion of liposomes to membranes. 22. 22. The method of claim 21, wherein the liposome comprises a bioactive agent. 23. 22. The method of claim 21, wherein the second lipid bilayer is the plasma membrane of the cell. 24. 24. The method of claim 23, wherein the cells are mammalian cells. 25. (A) preparing the liposomes represented by (i) and (ii) in an aqueous solution, (i) the liposome is (1) a physiologically active agent, (2) a neutral bilayer-forming lipid as well as being capable of being protonated. An outermost lipid bilayer comprising an ionizable lipid in a fusion promoting effective amount having a cation head group and an unsaturated acyl chain, and (3) a compartment adjacent to the outermost lipid bilayer containing an aqueous solution. and de, (ii) the pH of the aqueous solution is less than _the pK _a of lipids capable of ionizing in the outermost layer lipid bilayer, the pH of the outer aqueous medium (b) liposomes, outermost lipid bilayer higher than _the pK _a of the lipid ionizable in, thereby, there is a pH gradient across the outermost lipid bilayer and whereby the lipid ionizable inner monolayer of the outermost layer lipid bilayer Stored in the membrane (C) lowering the pH gradient when fusion of the liposome to the second lipid bilayer occurs, and (d) contacting the cell with the liposome such that the liposome fuses with the cell, The method of introducing a physiologically active agent into a cell, comprising: